The primary purpose of this proposal is to identify the effects of drugs of abuse (i.e. opioids) on glial cell mechanisms that contribute to drug-induced hyperalgesia and tolerance. These studies may also aid in elucidating substance abuse-induced plasticity, the physiological correlate to craving and relapse in drug addiction. Once thought to only serve a supportive role in the CNS, glial cells (specifically microglia and astrocytes) are now coming under intense investigation as major contributors to the pathology of many disorders including neuropathic pain, opioid tolerance and most recently, drug addiction. Following stress to the CNS (e.g. injury or xenobiotic administration) microglia become reactive as defined by enhanced surface or cytosolic protein expression, morphological changes, proliferation and migration. It has been proposed that once activated, specific microglia remain as postactivated or primed, i.e. they retain a memory to alter responses to subsequent stimulation. This could be an underlying mechanism for opioid tolerance following chronic administration, as well as drug addiction. We propose to study the role of microglia in opioid tolerance through investigation of the following hypothesis: Chronic opioids induce sustained CNS microglial reactivity leading to direct signaling between microglia and nociceptive neurons which manifests as hyperalgesia and/or tolerance. Further, we hypothesize that this selective activation potentiates extracellular ATP/ADP activation of P2X4 receptors initiating microglial migration. These migrating cells produce locally elevated chemokines (i.e. Monocyte Chemoattractant Protein-1/CCL2) and other diffusible proinflammatory factors, inducing dorsal horn neuron sensitization and tolerance formation. The overall hypothesis will be investigated using in vitro primary neonatal cortical and adult spinal microglia, in vivo adult rats and through the completion of the following Specific Aims: 1) Build on preliminary data to characterize opioid-induced microglial reactivity in vitro. 2) Investigate the mechanism of opioid-induced migration in vitro and in vivo. 3) Determine whether postactivated microglia retain memory and mechanisms to alter responses to subsequent exposure to opioids or stressors. Elucidating the mechanisms of opioid tolerance has the potential to aid a large patient population not served by current drugs by providing new targets for drug discovery. Taken together, these studies will provide a comprehensive and multidisciplinary approach to investigate the influence of CNS neuroimmune activation, specifically microglial reactivity on opioid tolerance and opioid-induced hyperalgesia. The data from these proposed studies will provide important new information and may culminate in novel pharmacopeia to reduce opioid tolerance. In addition, the results may have a more far-reaching impact on understanding CNS plasticity that occurs during opioid drug addiction. PUBLIC HEALTH RELEVANCE: The primary purpose of this proposal is to identify the effects of drugs of abuse (i.e. opioids) on glial cell mechanisms that contribute to drug-induced hyperalgesia and tolerance. These studies may also aid in elucidating substance abuse-induced plasticity, the physiological correlate to craving and relapse in drug addiction.